Method of controlling stress-related disease in livestock by administration of human IL-2

ABSTRACT

Disclosed herein is a method of mitigating stress-related syndromes in livestock, such as shipping fever in cattle, by administration of human IL-2.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending U.S. patentapplication Ser. No. 778,371, filed Sept. 20, 1985, now abandoned.

DESCRIPTION Technical Field

The invention relates to the prevention and amelioration in animals ofsymptoms of the malaise associated with livestock animals on feedlots, asymptomatology commonly known as "shipping fever". More particularly,the invention relates to a method of preventing or moderating thedisease-potentiating effects of stress and viral infection in animals byadministration of human interleukin-2 (hIL-2), including recombinantlyproduced hIL-2.

Background

Livestock food animals, particularly cattle, are adversely affected byshipment and feedlot conditions, which involve stress from overcrowding,weaning, transport, sometimes severe weather, and, in general, anon-natural environment. One syndrome, commonly known as "shippingfever", is sometimes also designated "bovine respiratory diseasesyndrome", or BRDS. It is a complex of disease symptoms rather than aspecific disease, and is characterized by immune suppression andpropensity to succumb to infection by one or more viral, or bacterialpathogens.

Other animals are also subject to adverse reactions to stress. Forexample, pigs, while ordinarily not shipped in the manner of cattle, cansuffer negative respiratory reactions to weaning or just to poorweather. Again, the symptomatology does not lend itself to experimentalmodels. No general treatment for stress-related disorders in livestockhas been found. Sick animals are typically treated with antibiotics.Recently several commercial entities have offered interferonpreparations for treating shipping fever.

There is considerable background information available with respect tothe biological activity of hIL-2. IL-2 is obtained from the supernatantof concanavlin-A (ConA) stimulated spleen cells or, presently, usingrecombinant technology, and has several measurable activities in vitro.First, it is a T-cell growth factor as measured by, for example,thymidine uptake when IL-2 is added to cultures of cytotoxic or helperT-cell lines. It is mitogenic with respect to adult thymocytes, andstimulates a cytotoxic cell response (e.g., lymphokine-activated-killer(LAK) cell). It has also been shown to replace helper T-cells in athymicmurine spleen cell cultures (Watson, J., et al, Immunological Rev (1980)51:257-278). Specifically, in the presence of IL-2 and antigen, specificT helper cells are generated which are able to contribute to antibodyresponses. Presumably this occurs because IL-2 is involved in theantigen-dependent maturation of helper T-cells in these nude mousespleen cultures.

IL-2 has also been shown to directly affect B cells in vitro. Bothproliferation and IgM and IgG secretion are enhanced by IL-2 inpopulations of purified, activated B cells (Mingari, M.C., et al.,Nature (1984) 312:641; Mittler, R., et al., J. Immunol. (1985) 134:2393-2399; Muraguchi, A., et al., J. Exp. Med. (1985) 161:181-197).

How these in vitro activities translate into a specific in vivomechanism for mounting an immune defense is not clear. However, withrespect to such in vitro studies, cross-reactivity among species ofvarious IL-2s has been studied. For example, Redelman, D., et al, JImmunol Meth (1983) 56:359-370) show that hIL-2 supports activated Tlymphocytes derived from rabbit and mouse to approximately the sameextent as they are supported by the endogenous forms of IL-2. Ruscetti,F. W., et al, Blood (1981) 57:379-393 were the first to demonstrate theability of hIL-2 to behave as a growth factor, not only for humanT-cells, but also peripheral blood lymphocytes or splenocytes from otherprimates, horse, guinea pig, cat, rat, and mouse. Carter, J., et al (FedProc (1985) 44:1290) disclose the ability of hIL-2 to enhance thedevelopment and maintenance of bovine cytotoxic lymphocytes in vitro.

Doyle, M. V., et al, J Bio Resp Mod (1985) 4:96-109 reports in vitrolymphocyte proliferation studies that compared the activities of nativehIL-2 and a recombinant form of IL-2 on human and animal lymphocytes.The native IL-2 and recombinant IL-2 exhibited the same range ofactivity on animal cells.

Some in vivo data are also available. The activity of IL-2 in vivo hasbeen shown to restore immunocompetence in nude mice in response toheterologous erythrocytes (Stotter, H., et al. Eur J Immunol (1980)10:719-722). There is some information concerning cross-speciesreactivity, as well. Reed, S. G., et al, J Immunol (1984) 133:3333,disclosed the ability of hIL-2 to reconstitute spleen cell responses inmice infected with a parasitic protozoan, and Farrar, J. J., et al,Immunol Rev (1982) 63:158, showed that in vivo injection of IL-2 ofhuman origin stimulates the splenic T-cells in nude mice.

In summary, it is known that IL-2 behaves in some manner in vivo tomediate a successful immune response, including a response to a specificantigen, and in vitro studies have shown that cross-species reactivityof hIL-2 is very diverse (prior in vivo crossspecies studies haveinvolved only murine subjects for hIL-2). However, because the mechanismof involvement of IL-2 in the immune response is not understood, it isnot possible to predict the behavior of IL-2 in boosting an immuneresponse to prevent or ameliorate a particular disease or to predict itsoverall effect. Accordingly, there is no suggestion in the art thatIL-2, and in particular hIL-2, would successfully mitigate the incidenceof shipping fever or other stressrelated syndromes that affectlivestock. This is the contribution of the present invention.

DISCLOSURE OF THE INVENTION

The invention provides a practical approach to controlling a poorlydefined disease that affects an estimated 12 million cattle per year inthe United States alone, resulting in a half million deaths among youngcattle with the concomitant waste in food supplies. The symptomatologyassociated with shipping and feedlot cultivation of these cattle can becontrolled using hIL-2, including the recombinant forms thereof. Inaddition, all livestock suffer from characteristic adverseinfection-related reactions to stress, and exhibit poorly definedsymptomatologies which are similarly treatable. By utilizing theavailable recombinant forms, a supply of effective hIL-2 is madeavailable in practical amounts and at relatively low cost.

In one aspect, the invention relates to methods of controlling (i.e.,prophylaxis or amelioration of severity or duration) shipping fever orother adverse reactions to stress in livestock by administration of aneffective dose of hIL-2, including that recombinantly produced. In otheraspects, the invention relates to symptomatolytic formulations of hIL-2for controlling such stress-related symptoms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of native hIL-2.

FIGS. 2A and 2B are dose-response curves showing the results of thelymphocyte proliferation tests described in section C.1 of the examples,infra.

FIG. 3 shows the effect of hIL-2 on blastogenesis of bovine and porcineT-lymphocytes.

MODES OF CARRYING OUT THE INVENTION

A. Definitions

As used herein, "hIL-2" refers to a polypeptide exhibiting the spectrumof activities characterizing this protein. Specifically, the proteinmust be capable of stimulating the proliferation of hIL-2 dependentcytolytic and helper T cell lines, as set forth in the standard assaysof Gillis, S, et al, J Immunol (1978) 120:2027-2032 and of Watson, J. JExp Med (1979) 150:1510-1519. The amino acid sequence of native hIL-2 isshown in FIG. 1. This primary amino acid sequence may be obtained as thenative protein from natural sources or may be recombinantly derived.Other primary sequences of modest modification including deletion,addition, substitution or alterations of the amino acids of the sequenceshown, which do not result in serious impairment of activity are, ofcourse, included in the definition. For example, it is established thatreplacement of the cysteine at position 125 with a neutral amino acidresults in a mutein of superior stability and satisfactory reactivity.(See U.S. Pat. No. 4,518,584; Doyle, M.V., et al, supra.)

In addition, IL-2, like any other protein, may exist in neutral or insalt form, and may contain associated non-protein materials in thenature of glycosylation, phosphorylation, or acetylation. Thesemodifications, too, are included in the definition so long as biologicalactivity is not destroyed thereby.

As used herein the term "stress-induced syndrome" refers to a state ofimmunosuppression in which an animal has a propensity to succumb toinfection by one or more bacterial or viral pathogens, lose weight, orexhibit general ill health.

"Shipping fever" or "bovine respiratory disease syndrome" (BRDS) isdefined as negative symptomatology including depression,immunosuppression, weight loss, respiratory problems, viral or bacterialinfection, and general ill health and death which are associated withthe transportation of cattle to, and the maintenance of cattle on,feedlots. The disease is defined in terms of epidemiology rather than interms of a model which describes the course of an infection or specificset of metabolic parameters. The criterion for effectiveness againstthis disease is the maintenance of healthy animals faced with thespecific conditions associated with shipping stress and feedlotmaintenance.

However, certain parameters of the disease are recognized. It ischaracterized by an abrupt onset, usually within two weeks of stress,and the symptoms may include dyspnea, cough, ocular and nasal discharge,inappetance and rapid weight loss, fever, increased lung sounds, andgeneral depression. Various bacteria and viral cultures have beenisolated from affected animals, including Pasteurella spp. Haemophilusspp. infectious bovine rhinotracheitis, parainfluenza-3 virus, andbovine respiratory syncytial virus. The disease typically affects 40-50%of exposed animals and the resulting deaths are typically 2-5% of theexposed population.

B. General Method

The formulations of the invention are most conveniently administered byintramuscular injections or as sustained release compositions althoughother methods of administration are possible. Specific formulations toprevent hydrolysis during digestion would be necessitated for oralformulation, and intravenous injections are generally uneconomic due tothe skill level and care required in the administration. Therefore,formulations suitable for intramuscular injection, expecially sustainedrelease formulations, are preferred.

Standard formulations are either liquid injectables or solids which canbe taken up in suitable liquids as suspensions or solutions forinjection. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, and so forth. Nontoxic auxiliarysubstances, such as wetting agents, buffers, or emulsifiers may also beadded. One specific useful formulation contains an effective amount ofdetergent, such as 0.1% sodium dodecyl sulfate (SDS), to effectsolubility and bacteriostasis. A variety of techniques may be used toeffect long-term stability and slow release. For example, stability isenhanced by coupling to a homopolymer such as polyethylene glycol (PEG).As described in copending commonly owned U.S. patent application Ser.No. 749,955, filed June 26, 1985, and incorporated herein by reference,proteins . . . are covalently bonded via one or two of the amino acidresidues of the proteins, depending mainly on the reaction conditionsand the particular protein employed. While the residues may be anyreactive amino acids on the portein, such as one or two cysteines withreactive thiol groups to form a thiol ester linkage or N-terminal aminoacid groups such as proline on IL-2, which proline may react when higherlevels of modification are employed, preferably the reactive amino acidis lysine, which is linked to the reactive group of the activatedpolymer through its free ε-amino group.

The homopolymer to which the protein is attached is a homopolymer ofpolyethylene glycol (PEG) or polypropylene glycol (PPG), each of whichmust have a molecular weight between about 500 and 20,000, preferablybetween 2,000 and 10,000, depending on the particular protein employed.In addition, the homopolymer must have a straight chain so as not torender the protein immunogenic. Therefore, preferably the homopolymer isunsubstituted, but it may also be substituted at one end with an alkylgroup containing one carbon atom in its chain. This signifies that oneterminal OH group of the glycol is replaced by an O-alkyl group.Preferably the alkyl group is a C₁ -C₄ alkyl group, and most preferablya methyl group, to prevent crosslinking reactions. Most preferably, thehomopolymer is monomethyl-substituted PEG with a molecular weight of2,000 to 10,000.

The IL-2 is linked to the homopolymer via a coupling agent, which musthave reactive groups which will selectively react with free amino orother reactive groups on the protein. For IL-2 the amount of homopolymeremployed is preferably no more than 50 moles per mole of IL-2, and mostpreferably is about 5 to 20 moles per mole of IL-2, depending on thespecific properties ultimately desired, i.e., the final amount is abalance to maintain optimum activity, while at the same time maximizing,if possible, the half-life of the protein. Preferably, at least about50% of the biological activity of the protein is retained, and mostpreferably 100% is retained.

The covalent coupling reaction may take place by any suitable methodgenerally used for coupling between biologically active materials andinert polymers, provided that it take place at a pH of 8 to 10,preferably about 9.0, if the reactive groups on the protein are lysinegroups. Use of pH values lower than 8 for the lysine reaction may resultin decreased modification of the protein. Generally the process involvesreacting at least one terminal hydroxyl group of the homopolymer with acoupling agent to provide an activated polymer, and thereafter reactingthe protein with the activated polymer to produce the solubilizedprotein suitable for formulation.

The above coupling reaction can be performed by several methods, whichmay involve one or more steps. Examples of suitable coupling agentswhich can be used in a one-step reaction include cyanuric acid chloride(2,4,6-trichloro-S-triazine) and cyanuric acid fluoride.

In a preferred embodiment the reaction takes place in two steps whereinthe homopolymer is reacted first with an acid anhydride such as succinicor glutaric anhydride to form a carboxylic acid, and the carboxylic acidis then reacted with a compound capable of reacting with the carboxylicacid to form an activated polymer with a reactive ester group which iscapable of reacting with the protein. Examples of such compounds includeN-hydroxysuccinimide, 4-hydroxy-3-nitrobenzene sulfonic acid, and thelike, and preferably N-hydroxysuccinimide or 4-hydroxy-3-nitrobenzenesulfonic acid is used. For example, monomethyl substituted PEG may bereacted in the presence of a mild base with succinic anhydride. Themonomethyl PEG-succinic acid thus produced is then reacted withN-hydroxysuccinimide in the presence of a carbodiimide reagent such asdicyclohexyl or isopropyl carboiimide to produce the activated polymer,methoxypolyethylene glycolyl-N-succinimidyl succinate, which can then bereacted with the protein. This method is described in detail inAbuchowski et al., Cancer Biochem. Biophys., 7, 175-186 (1984). Inanother example the monomethyl substituted PEG may be reacted withglutaric anhydride followed by reaction with 4-hydroxy-3-nitrobenzenesulfonic acid (HNSA) in the presence of dicyclohexyl carbodiimide toproduce the activated polymer. HNSA is described in Bhatnagar et al.,Peptides: Synthesis-Structure-Function, Proceedings of the SeventhAmerican Peptide Symposium, Rich, et al. (eds.) (Pierce Chemical Co.,Rockford IL, 1981), p. 97-100, and in Nitecki et al., High-TechnologyRooute to Virus Vaccines (American Society for Microbiology, anticipatedFall 1985--in press), entitled "Novel Agent for Coupling SyntheticPeptides to Carriers and Its Application."

This PEG-hIL-2 complex, called "PEGylated" hIL-2, is particularly usefulfor administering a single sustained action dose of hIL-2.

As described in U.S. patent application Ser. No. 749,955, the PEGylatedhIL-2 complex is formed by covalent bonding of the IL-2 via one or twoamino acid residues. While the residues may be any reactive amino acidson the protein, such as one or two cysteines with reactive thiol groupsto form a thiol ester linkage or N-terminal amino acid groups, thereactive amino acid is preferably lysine, which is linked topolyethylene glycol through its free epsilon-amino group.

The polyethylene glycol to which the IL-2 is coupled has a molecularweight between about 500 and 20,000, preferably between 2,000 and10,000. In addition, the PEG must have a straight chain so as not torender the protein immunogenic. Thus, the PEG is preferablyunsubstituted, but may be substituted at one end with an alkyl groupcontaining one carbon atom in its chain. This signifies that oneterminal OH group of the glycol is replaced by an O-alkyl group.Preferably the alkyl group is a C₁ -C₄ alkyl group, and most preferablya methyl group, to prevent crosslinking reactions. Most preferably, thePEG is monomethyl-substituted PEG with a molecular weight of 2,000 to10,000.

The IL-2 is linked to the PEG via a coupling agent, which must havereactive groups which will selectively react with free amino or otherreactive groups on the protein. It is recommended that generally theamount of PEG employed is preferably no more than 50 moles per mole ofIL-2, and most preferably is about 5 to 20 moles per mole of IL-2,depending on the specific properties ultimately desired, i.e. the finalamount is a balance to maintain optimum activity, while at the same timemaximizing, if possible, the half-life of the IL-2. Preferably, at leastabout 50% of the biological activity of the IL-2 is retained, and mostpreferably 100% is retained.

The covalent coupling reaction may take place by any suitable methodgenerally used for coupling between biologically active materials andinert polymers, provided that it takes place at a pH of 8 to 10,preferably about 9.0, if the reactive groups on the protein are lysinegroups. Use of pH values lower than 8 for the lysine reaction may resultin decreased modification of the IL-2. Generally the process involvesreacting at least one terminal hydroxyl group of the PEG with a couplingagent to provide an activated polymer, and thereafter reacting the IL-2with the activated polymer to produce the solubilized protein suitablefor formulation.

The above coupling reaction can be performed by several methods, whichmay involve one or more steps. Examples of suitable coupling agentswhich can be used in a one-step reaction include cyanuric acid chloride(2,4,6-traichloro-S-triazine) and cyanuric acid fluoride.

In a preferred embodiment the reaction takes place in two steps whereinthe PEG is reacted first with an acid anhydride such as succinic orglutaric anhydride to form a carboxylic acid, and the carboxylic acid isthen reacted with a compound capable of reacting with the carboxylicacid to form an activated polymer with a reactive ester group which iscapable of reacting with the protein. Examples of such compounds includeN-hydroxysuccinimide, 4-hydroxy-3-nitrobenzene sulfonic acid, and thelike, and preferably N-hydroxysuccinimide or 4-hydroxy-3-nitrobenzene isused. For example, monomethyl substituted PEG may be reacted in thepresence of a mild base with succinic anhydride. The monomethylPEG-succinic acid thus produced is then reacted withN-hydroxysuccinimide in the presence of a carbodiimide reagent such asdicyclohexyl or isopropyl carbodiimide to produce the active polymer,methoxypolyethylene glycolyl-N-succinimidyl succinate, which can then bereacted with the protein. This method is described in detail inAbuchowski et al., Cancer Biochem. Biophys., 7, 175-186 (1984). Inanother example the monomethyl substituted PEG may be reacted withglutaric anhydride followed by reaction with 4-hydroxy-3-nitrobenzenesulfonic acid (HNSA) in the presence of dicyclohexyl carbodiimide toproduce the activated polymer. HNSA is described in Bhatnagar et al.,Peptides: Synthesis-Structure-Function, Proceedings of the SeventhAmerican Peptide Symposium, Rich et al. (eds.) (Pierce Chemical Co.,Rockford, IL 1981), pp. 97-100, and in Nitechki et al., High TechnologyRoute to Virus Vaccines (American Society for Microbiology, 1985),entitled "Novel Agent for Coupling Synthetic Peptides to Carriers andIts Application").

Sustained and continuous release formulations are of considerablevariety, as is understood by those skilled in the art. An exemplarycomposition for sustained release parenteral administration is aninjectable microcapsule formulation that with a single injection willdeliver recombinant hIL-2 or soluble forms of hIL-2, such as PEGylatedhIL-2, at a controlled rate of about 10³ to 10⁵ units/kg/day for aduration of 14 to 30 days. (Pure hIL-2 has a specific activity of about3-6×10⁶ u/mg.) The microcapsule formulation is a free-flowing powderconsisting of spherical particles 20 to 100 μm in diameter that can beinjected intramuscularly or subcutaneously with a conventionalhypodermic needle, and the microcapsules consist of 0.5 to 5% hIL-2encapsulated in poly(DL-lactide-co-glyco lide) (DL-PLG) excipient, abiodegradable, biocompatible polyester. Alternative standardformulations for sustained release are also usable.

The regime of administration for shipping fever will depend on theconditions of shipment and the feedlot. It is preferred thatadministration be continuous and be begun prior to shipment or at leastas early as arrival on the feedlot and be continued over a period of,for example, 14-30 or more days. The term "continuous" is intended todenote true continuous administration, such as is achieved via asustained release dosage form as well as a multiplicity of intermittentadministrations of hIL-2 (or enhanced half-life forms of hIL-2 such asPEGylated hIL-2) that provide a pharmacokinetic pattern that mimics thatachieved by true continuous administration. Daily doses in the range ofabove about 10³ and below about 10⁶ units/kg/day, preferably about 10⁴to 10⁵ units/kg/day, are generally used. In cattle, doses above about10⁶ units/kg/day began to cause undesirable side effects.

For other livestock stress-induced or respiratory distress syndromes,the regime and amounts administered will depend on the nature and sizeof the animal (e.g., pig, goat, sheep, etc.) and on the severity of thesymptoms. It is likely, however, that the effective dose for suchsyndromes will be in the same (on a unit weight basis) range as thatused for shipping fever.

The hIL-2 may be administered by itself or as a supplement to vaccinesused to protect against stress-related diseases.

C. Examples

The following examples are intended to further support or illustrate butnot to limit the invention.

C.1. In Vitro Activity

In vitro activity with respect to bovine and porcine peripheral bloodmononuclear cells (PBMC) has been shown for recombinant hIL-2 (Fong,Susan, et al, Vet Immunol and Immunopathol (1986) 11:91-100 andincorporated herein by reference). The hIL-2 used in this work isdesignated des-alanyl-rIL-2_(ser125), lacks an initial alanine, and hasa serine rather than a cysteine at position 125. It was shown to bemitogenic for unactivated bovine and porcine PBMC, and to be able tomaintain the long-term growth of ConA-activated PBMC from both species.FIGS. 2A and 2B are curves showing the dose-response of ConA-activatedbovine (2A) and porcine (2B) PBMC to des-alanyl-rIL-2_(ser125). Also,bovine and porcine PBMC preincubated with des-alanyl-rIL-2_(ser125) for1-5 days showed enhanced cytotoxicity against tumor cell targets.

In addition, Stott, J. L., et al (submitted and incorporated herein byreference) have shown that bovine and porcine peripheral bloodlymphocytes were responsive to human recombinant IL-2 in lymphocyteblastogenesis assays. Blastogenesis was determined by incorporation of ³H-thymidine (18 hr pulse) in 4-day lymphocyte cultures, and the resultsexpressed as the log₁₀ of the geometric mean (G_(x)) of disintegrationsper minute (DPM)/culture and plotted by nonlinear regression analysis asshown in FIG. 3. Mitogen dilution and concentration of hIL-2 in unitsare shown on the X-axis. These results show that the effect of hIL-2 onbovine and porcine cells is comparable to that shown by the plantlectins PHA and ConA, which are known to stimulate blastogenesis.

C.2. Potentiation of Cell-Mediated Immunity

Since respiratory diseases are predominantly controlled by the cellular(T-cell) immune system, the ability of hIL-2 to boost the cellularimmune response in livestock is indicative of its effectiveness againstthese symptomologies. In vivo injections of recombinant hIL-2 producedelevated levels of lymphocyte blastogenesis in the blood of calves.

Specifically, eight calves weighing 135-225 kg (3-5 months old) wererandomly sorted into 4 groups of 2 each which received weekly injectionsfor one month as follows: Groups 1, 2, and 3 received 10⁴, 10⁵, and 10⁶units/kg, respectively, intramuscularly; group 4 received onlyexcipient. The animals were assessed for lymphocyte stimulation. Theresults show that resting lymphocyte activity was elevated by therecombinant hIL-2 treatment as determined by blastogenesis assaysperformed prior to each inoculation over the period in calves receiving10⁵ and 10⁶ units/kg only. For calves receiving 10⁵ units/kg, lymphocyteactivity returned to normal within two weeks following the last IL-2administration; 10⁶ units/kg-injected calves remained elevated at thattime.

C.3. Treatment of Shipping Fever

Two hundred heifers were purchased from several different sources inTennessee and transported to a research feedlot in Colorado. The averageweight of the animals was approximately 400 lbs. The animals weresegregated randomly (weight and breed) into four groups, designated Ithrough IV.

Recombinant hIL-2 (des-alanyl-rIL-2_(ser125)) was formulated in 0.05%SDS and administered intramuscularly to the animals upon entry to thefeedlot. All animals were treated daily, five times per week, for twoweeks. The dose protocols for the four groups were as follows.

    ______________________________________                                        Group            IL-2 Dose (u/kg/day)                                         ______________________________________                                        I                2 × 10.sup.4 (high dose)                               II               2 × 10.sup.3 (mid dose)                                III              2 × 10.sup.2 (low dose)                                IV               control (diluent)                                            ______________________________________                                    

The animals did not receive standard BRDS-related vaccination. Theywere, by chance, subjected to severe snow and cold weather during theirfirst days on the feedlot, and accordingly, were placed on silage feedearly on. The health of the animals was observed on a daily basis bypersonnel blind to experimental treatment. The animals were weighed atregular intervals. Table 1 reports the results of the treatment as ofday 21.

                  TABLE 1                                                         ______________________________________                                        Mortality                                                                               Number Dead/Total                                                   ______________________________________                                        Control  21/50                                                                Low Dose 20/50                p = 0.839                                       Mid Dose 26/50                p = 0.316                                       High Dose                                                                              14/50                p = 0.142                                       Incidence of Disease                                                                    Number Sick or Dead/Total                                           ______________________________________                                        Control  43/50                                                                Low Dose 42/50                p = 0.779                                       Mid Dose 43/50                p = 1.000                                       High Dose                                                                              38/50                P = 0.202                                       Severity of Disease                                                                    Average Daily Severity Score of                                               Group (Score 0-3; Death = 4)                                         ______________________________________                                        Control  1.76                                                                 Low Dose 1.79                 p = 0.950                                       Mid Dose 1.93                 p = 0.395                                       High Dose                                                                              1.38                 p = 0.052                                       ______________________________________                                    

Morbidity and mortality rates during the study were higher thanexpected. As reported some groups showed 85% morbidity and 50%mortality. Sickness was observed as early as two days into the study.Several factors may have been responsible for the extreme severity ofBRDS seen in this study: the severe snow and cold weather; the animalswere `light-weight` (400 lbs avg) and `thin-skinned` (from Tennessee);groups had been `put-together` from several sources (thus, they were not`fresh` and many had seen several salebarns prior to shipping toColorado); and the animals were placed on silage feed early on, and mayhave been eating poorly.

In the clinical judgement of the personnel observing the health of theanimals, the high-dose IL-2 group consistently "looked better". This issupported by the data in Table 1 in which the high-dose IL-2 groupshowed a consistent trend towards decreased mortality; decreasedincidence of disease; and decreased severity of disease.

In all cases, the high-dose group performed better than the controlgroup. Although the statistical significance of these differences(p-value), is marginal (using the strict definition of p<0.05), allresults are consistent.

Additional measures not presented in Table 1 also supported the trendtoward efficacy in the highdose IL-2 group. For instance, animals in thehigh-dose group which died, did so later in the study than did controlanimals.

As of day 21, there were no differences in the average weight ofsurviving animals. There were, however, significant differences in thetotal pay-weight per group, since more animals survived in the high-dosegroup.

Modifications of the above-described modes for carrying out theinvention that are obvious to those of skill in the fields of veterinarymedicine, immunology, pharmacology, and related fields are intended tobe within the scope of the following claims.

We claim:
 1. A method of diminishing the incidence of shipping fever inlivestock which comprises administering to subject livestock an amountof a water soluble form of hIL-2 in the range of above about 10³ andbelow about 10⁶ u/kg/day over a period of from about 14 to 30 days,wherein the water soluble form of hIL-2 is hIL-2 covalently bondedthrough one or two amino acids via a coupling agent to polyethyleneglycol having a molecular weight in the range of from about 500 to200,000, and wherein no more than about 50 moles of polyethylene glycolare employed per mole hIL-2.
 2. A method of diminishing the incidence ofshipping fever in livestock which comprises administering to subjectlivestock an amount of a water soluble form of des-alanyl-rIL-2_(ser125)in the range of above about 10³ and below about 10⁶ u/kg/day over aperiod of from about 14 to 30 days, wherein the water soluble form ofhIL-2 is hIL-2 covalently bonded through one or two amino acids via acoupling agent to polyethylene glycol having a molecular weight in therange of from about 500 to 200,000, and wherein no more than about 50moles of polyethylene glycol are employed per moledes-alanyl-rIL-2_(ser125).
 3. A method of ameliorating the symptoms ofshipping fever in livestock which comprises administering to subjectlivestock an amount of a water soluble form of hIL-2 in the range ofabove about 10³ and below about 10⁶ u/kg/day over a period of from about14 to 30 days, wherein the water soluble form of hIL-2 is covalentlybonded through one or two amino acids via a coupling agent topolyethylene glycol having a molecular weight in the range of from about500 to 200,000, and wherein no more than about 50 moles of polyethyleneglycol are employed per mole hIL-2.
 4. A method of ameliorating thesymptoms of shipping fever in livestock which comprises administering tosubject livestock an amount of a water soluble form ofdes-analy-rIL-2_(ser125) in the range of above about 10³ and below about10⁶ u/kg/day over a period of from about 14 to 30 days, wherein thewater soluble form of hIL-2 is hIL-2 covalently bonded through one ortwo amino acids via a coupling agent to polyethylene glycol having amolecular weight in the range of from about 500 to 200,000, and whereinno more than about 50 moles of polyethylene glycol are employed per moledes-alanyl-rIL-2_(ser125).
 5. A method of shortening the duration ofshipping fever in livestock which comprises administering to subjectlivestock an amount of a water soluble form of hIL-2 in the range ofabove about 10³ and below about 10⁶ u/kg/day over a period of from about14 to 30 days, wherein the water soluble form of hIL-2 is covalentlybonded through one or two amino acids via a coupling agent topolyethylene glycol having a molecular weight in the range of from about500 to 200,000, and wherein no more than about 50 moles of polyethyleneglycol are employed per mole hIL-2.
 6. A method of shortening theduration of shipping fever in livestock which comprises administering tosubject livestock an amount of a water soluble form ofdes-alanyl-rIL-2_(ser125) in the range of above about 10³ and belowabout 10⁶ u/kg/day over a period of from about 14 to 30 days, whereinthe water soluble form of hIL-2 is hIL-2 covalently bonded through oneor two amino acids via a coupling agent to polyethylene glycol having amolecular weight in the range of from about 500 to 200,000, and whereinno more than about 50 moles of polyethylene glycol are employed per moledes-alanyl-rIL-2_(ser125).